Novel glucose-6-phosphate dehydrogenase

ABSTRACT

The present invention relates to a novel glucose-6-phosphate dehydrogenase (hereinafter referred to as “G6PD”) derived from a bacterium belonging to the genus Corynebacterium, a DNA encoding the enzyme, a recombinant DNA comprising the DNA, a transformant comprising the recombinant DNA, a transformant comprising the DNA on its chromosome, and a process for producing L-amino acid or G6PD which comprises culturing the transformant.  
     According to the present invention, a modified G6PD and a DNA encoding the G6PD are obtained, and the productivity of L-amino acid by a microorganism can be improved by using the modified G6PD.

TECHNICAL FIELD

[0001] The present invention relates to a novel glucose-6-phosphatedehydrogenase (hereinafter referred to as “G6PD”) derived from abacterium belonging to the genus Corynebacterium, a DNA encoding theenzyme, a recombinant DNA comprising the DNA, a transformant comprisingthe recombinant DNA, a transformant comprising the DNA on itschromosome, and a process for producing L-amino acid which comprisesculturing the transformant.

BACKGROUND ART

[0002] In order to obtain a bacterial strain which produces an aminoacid efficiently, it is important to know properties of genes relatingto the biosynthesis of the amino acid in the bacterium and their stylefor controlling expression and activity and to carry out rationalbreeding based thereon.

[0003] One of the important methods for understanding the functions ofgenes relating to the amino acid production is a genetic method, forexample, in which a relationship between increase or decrease in aminoacid productivity and gene mutation is clarified.

[0004] Breeding of amino acid-producing microorganisms is mainly carriedout by endowing resistance to drugs such as amino acid analogues and thelike, but in many cases, it is not clear which gene provides theproductivity improvement by its mutation.

[0005] NADPH is necessary as a coenzyme at reduction reaction in theamino acid biosynthesis in many microorganisms. For example, 4 moleculesof NADPH are necessary for the biosynthesis of 1 molecule of L-lysine.In the same manner, 3 molecules of NADPH are necessary for 1 molecule ofthreonine, and 5 molecules of NADPH are necessary for 1 molecule ofisoleucine. Thus, two or more molecules of NADPH are necessary for thebiosynthesis of 1 molecule of most amino acids. Accordingly, supply ofNADPH is an important subject in producing these amino acids usingmicroorganisms.

[0006] In many microorganisms, NADPH-supplying enzymes are limited. Itis considered that the enzymes which can supply NADPH on the mainpathways of sugar metabolism of the microorganisms are mainly G6PD [EC1.1.1.49] and 6-phosphogluconate dehydrogenase [EC 1.1.1.4] in thepentose phosphate pathway (HMP) and isocitrate dehydrogenase[EC-1.1.1.41] in the TCA pathway.

[0007] Particularly, G6PD, which is the first enzyme of HMP and is alsothe parting point-enzyme from the Embden-Meyerhof pathway (EMP), isconsidered to be a very important enzyme for the production of variousamino acids by bacteria belonging to the genus Escherichia and the genusCorynebacterium, and various analyses have been carried out mainly onits various biochemical properties. For example, G6PD of bacteriabelonging to the genus Corynebacterium is described in Journal ofBacteriology, 98, 1151 (1969), Agricultural and Biological Chemistry,51, 101 (1987) and Japanese Published Unexamined Patent Application No.224661/97, but the investigation for productivity improvement of aminoacids using the enzyme has not been reported.

[0008] Also, the nucleotide sequence of G6PD of bacteria such asEscherichia coli and Corynebacterium glutamicum, the nucleotide sequenceof the gene has been found (Journal of Bacteriology, 173, 968 (1991) andJapanese Published Unexamined Patent Application No. 224661/97), but theinvestigation for productivity improvement of amino acids using the genehas not been reported.

DISCLOSURE OF THE INVENTION

[0009] An object of the present invention is to produce L-amino acidindustrially advantageously by using G6PD relating to the biosynthesisof the L-amino acid, a DNA encoding the enzyme, a recombinant DNAobtained by inserting the DNA into a vector or a transformant comprisingthe recombinant DNA to thereby further increase the L-amino acidproductivity by a microorganism.

[0010] The present inventors have succeeded in isolating a DNA encodinga polypeptide comprising the amino acid sequence represented by SEQ IDNO:2, and found that it can be used in the production of L-amino acid.Also, as a result of intensive studies, the present inventors found thata polypeptide in which Ala at position 213 in the amino acid sequencerepresented by SEQ ID NO:2 is replaced with an other amino acid andwhich has the G6PD activity can further improve the productivity ofL-amino acid. Thus, the present invention has been accomplished.Specifically, the present invention relates to the following (1) to(21).

[0011] (1) A polypeptide which comprises the amino acid sequencerepresented by SEQ ID NO:2.

[0012] (2) A polypeptide which comprises an amino acid sequence in whichAla at position 213 in the amino acid sequence represented by SEQ IDNO:2 is replaced with an other amino acid, and has G6PD activity.

[0013] (3) A polypeptide which comprises the amino acid sequencerepresented by SEQ ID NO:12.

[0014] (4) A polypeptide which comprises an amino acid sequence in whichone or several amino acids other than the amino acid residue at position213 in the amino acid sequence of the polypeptide according to (2) aredeleted, substituted or added, and has G6PD activity.

[0015] (5) A polypeptide which comprises an amino acid sequence in whichone or several amino acids other than the amino acid residue at position213 in the amino acid sequence represented by SEQ ID NO:12 are deleted,substituted or added, and has G6PD activity.

[0016] (6) A DNA which encodes the polypeptides according to any one of(1) to (5).

[0017] (7) A DNA which comprises the nucleotide sequence represented bySEQ ID NO:1.

[0018] (8) A DNA which comprises a nucleotide sequence in which anucleotide sequence of positions 637 to 639 encoding Ala in thenucleotide sequence represented by SEQ ID NO:1 is replaced with a codonencoding an amino acid other than Ala.

[0019] (9) A DNA which comprises the nucleotide sequence represented bySEQ ID NO:11.

[0020] (10) A DNA which hybridizes with a DNA comprising the nucleotidesequence represented by SEQ ID NO:1 under stringent conditions, andencodes a polypeptide having glucose-6-phosphate dehydrogenase activity,wherein a nucleotide sequence corresponding to the nucleotide sequenceof positions 637 to 639 encoding Ala in the nucleotide sequencerepresented by SEQ ID NO:1 is replaced with a codon encoding an aminoacid other than Ala.

[0021] (11) A DNA which hybridizes with a DNA comprising the nucleotidesequence represented by SEQ ID NO:1 under stringent conditions, andencodes a polypeptide having G6PD activity, wherein a nucleotidesequence corresponding to the nucleotide of position 637 in thenucleotide sequence represented by SEQ ID NO:1 is replaced with adenine.

[0022] (12) A recombinant DNA which is obtainable by inserting the DNAaccording to any one of (6) to (11) into a vector.

[0023] (13) The recombinant DNA according to (12), wherein therecombinant DNA is replicable in a microorganism belonging to the genusEscherichia or the genus Corynebacterium.

[0024] (14) A plasmid pCRBzwfM comprised in Escherichia coli TOP10 (FERMBP-7135).

[0025] (15) A transformant which is obtainable by introducing therecombinant DNA or plasmid according to any one of (12) to (14) into ahost cell.

[0026] (16) The transformant according to (15), wherein the host cell isa microorganism which is capable of producing L-amino acid.

[0027] (17) The transformant according to (16), wherein the host cell isa microorganism belonging to the genus Escherichia or the genusCorynebacterium.

[0028] (18) A transformant belonging to the genus Escherichia or thegenus Corynebacterium, which comprises a chromosome into which the DNAaccording to any one of (6) to (11) is artificially integrated.

[0029] (19) The transformant according to (17) or (18), wherein themicroorganism belonging to the genus Corynebacterium is Corynebacteriumglutamicum.

[0030] (20) A process for producing a polypeptide, which comprisesculturing the transformant according to any one of (15) to (19) in amedium to form and accumulate the polypeptide according to any one of(1) to (5) in a culture, and recovering the polypeptide from theculture.

[0031] (21) A process for producing L-amino acid, which comprisesculturing the transformant according to any one of (16) to (19) in amedium to form and accumulate L-amino acid which is biosynthesized usingNADPH in the culture, and recovering the L-amino acid from the culture.

[0032] (22) The process for producing L-amino acid according to (21),wherein the L-amino acid which is biosynthesized using NADPH is selectedfrom L-lysine, L-threonine, L-isoleucine, L-tryptophan, L-phenylalanine,L-tyrosine, L-histidine and L-cysteine.

[0033] (23) The process for producing L-amino acid according to (21),wherein the L-amino acid is L-lysine.

[0034] The present invention is described below in detail.

[0035] The polypeptide of the present invention is a polypeptide whichcomprises the amino acid sequence represented by SEQ ID NO:2 or apolypeptide which comprises an amino acid sequence in which Ala atposition 213 of the amino acid sequence represented by SEQ ID NO:2 issubstituted with an other amino-acid and has G6PD activity. Examples ofthe polypeptide include a polypeptide comprising the amino acid sequencerepresented by SEQ ID NO:12.

[0036] A polypeptide which comprises an amino acid sequence in which oneor several amino acids in the amino acid sequence comprised in thepolypeptide are deleted, substituted or added is also included in thepolypeptide of the present invention, so long as it has G6PD activity.However, the polypeptide does not include known G6PD (for example,polypeptide in which Thr at position 120 in SEQ ID NO:2 is replaced withAla).

[0037] The protein which comprises an amino acid sequence in which oneor several amino acids are deleted, substituted or added and has G6PDactivity can be obtained by introducing a site-directed mutation into aDNA encoding a polypeptide comprising the amino acid sequencerepresented by SEQ ID NO:2 or 12, using the site-directed mutagenesisdescribed in Molecular Cloning, A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press (1989) (hereinafter referred to as“Molecular Cloning, Second Edition”), Current Protocols in MolecularBiology, John Wiley & Sons (1987-1997) (hereinafter referred to as“Current Protocols in Molecular Biology”), Nucleic Acids Research, 10,6487 (1982), Proc. Natl. acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315(1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. acad. Sci.USA, 82, 488 (1985) and the like. It also can be obtained by introducinga site-directed mutation according to the above method into a DNAencoding a polypeptide which originally has a sequence in which one orseveral amino acids are deleted, substituted or added from the aminoacid sequence represented by SEQ ID NO:2 and has the G6PD activity(e.g., a G6PD derived from a microorganism close to Corynebacteriumglutamicum) to thereby replace an amino acid corresponding to the aminoacid at position 213 of the amino acid sequence represented by SEQ IDNO:2 with an other amino acid.

[0038] The number of amino acids to be deleted, substituted or added isnot particularly limited, but is the number that can be deleted,substituted or added by a well known method such as the site-directedmutagenesis or the like, and is preferably from 1 to 10 and morepreferably from 1 to 5.

[0039] Also, in order that the polypeptide of the present invention hasthe G6PD activity, it is preferable that the polypeptide has homology ofat least 60% or more, generally 80% or more, particularly 95% or more,with the amino acid sequence described in SEQ ID NO:2 or 12, whencalculated using BLAST [J. Mol. Biol., 215, 403 (1990)], FASTA [Methodsin Enzymology, 183, 63-98 (1990)] or the like.

[0040] Examples of the DNA of the present invention encoding thepolypeptide of the present invention include a DNA comprising thenucleotide sequence represented by SEQ ID NO:1, a DNA comprising anucleotide sequence in which a nucleotide sequence of positions 637 to639 encoding Ala in the nucleotide sequence represented by SEQ ID NO:1is replaced with a codon encoding an amino acid other than Ala(hereinafter referred to as “SEQ ID NO:1 sub”), and a DNA comprising thenucleotide sequence represented by SEQ ID NO:11 in which the nucleotideat position 637 in the nucleotide sequence SEQ ID NO:1 is adenine.

[0041] The DNA of the present invention also includes a DNA whichhybridizes with a DNA comprising the nucleotide sequence represented bySEQ ID NO:1 under stringent conditions, has a nucleotide sequence inwhich a nucleotide sequence of positions 637 to 639 encoding Ala in thenucleotide sequence represented by SEQ ID NO:1 is replaced with a codonencoding an amino acid other than Ala, and encodes a polypeptide havingG6PD activity. However, the DNA of the present invention does notinclude known DNA (e.g., a DNA in which adenine at position 358 in SEQID NO:1 is replaced with guanine).

[0042] Herein, the DNA which hybridizes with the DNA of SEQ ID NO:1under stringent conditions means a DNA which is obtainable by colonyhybridization, plaque hybridization, Southern blot hybridization or thelike using a DNA comprising the nucleotide sequence represented by SEQID NO:1 or 11 as a probe, and examples thereof include a DNA which canbe identified by carrying out hybridization at 65° C. in the presence of0.7 to 1.0 mol/l of sodium chloride using a filter on which a colony- orplaque-derived DNA is immobilized, and then washing the filter at 65° C.using 0.1-fold to 2-fold concentration SSC solution (composition of1-fold concentration SSC contains 150 mmol/l sodium chloride and 15mmol/l sodium citrate). The hybridization can be carried out accordingto the method described in, e.g., Molecular Cloning Second Edition,Current Protocols in Molecular Biology or DNA Cloning 1: CoreTechniques, A Practical Approach, Second Edition, Oxford University(1995). Examples of the DNA which can be hybridized include a DNAcontaining a nucleotide sequence having at least 60% or more of identitywith the nucleotide sequence represented by SEQ ID NO:1 or 11,preferably a DNA containing a nucleotide sequence having 80% or more ofidentity, more preferably a DNA containing a nucleotide sequence having95% or more of identity, when calculated using the BLAST, FASTA or thelike.

[0043] The DNA of the present invention can be obtained fromCorynebacterium glutamicum No. 58 (FERM BP-7134) or from a mutant havingincreased L-amino acid productivity obtained by applying a generalmutagenizing operation to the strain.

[0044] Examples of the mutagenizing operation include the conventionalmethod using N-methyl-N′-nitro-N-nitrosoguanidine (NTG); (MicrobialExperimentation Manual, 1986, p. 131, Kodansha Scientific).

[0045] The DNA of the present invention can be isolated by the followingmethod.

[0046] That is, a chromosomal DNA is prepared from a strain containingthe DNA by, e.g., the method of Saito et al. [Biochimica et BiophysicaActa, 72, 619 (1963)], and the chromosomal DNA is digested with anappropriate restriction enzyme. The obtained DNA fragment is ligatedwith a vector (e.g., plasmid) which is autonomously replicable inbacterial cells, and the ligated DNA is introduced into a microorganismwhich is defective in the G6PD activity. A transformant is isolated fromthe obtained microorganism using the G6PD activity as the index, and thegene for the enzyme is isolated from the transformant.

[0047] For example, a strain of Escherichia coli which is defective inonly glucose-6-phosphate isomerase can grow in a medium containingglucose as the sole carbon source, but a strain further defective inG6PD cannot grow in a medium containing glucose as the sole carbonsource [Escherichia coli and Salmonella typhimurium, 192 (1996)]. Thus,the DNA of the present invention can be isolated from the strain byselecting a strain which became able to grow in a medium containingglucose as the sole carbon source from the strains obtained byintroducing the DNA into the double-defective strain.

[0048] The microorganism into which the DNA of the present invention isintroduced may be a bacterium belonging to any genus, so long as the DNAcan be expressed. Also, the autonomously replicable vector may be anyvector, so long as it can autonomously replicate in the bacterium. Forexample, when a microorganism belonging to the genus Escherichia,particularly Escherichia coli, is used, the autonomously replicablevector include pUC18 (manufactured by Takara Shuzo) and pBluescriptSK(−) (manufactured by TOYOBO). Also, it may be a shuttle vector whichis autonomously replicable in both Escherichia coil and a bacterium ofthe genus Corynebacterium, such as pCE54 (Japanese Published UnexaminedPatent Application No. 105999/83).

[0049] The vector can be ligated with the DNA of the present inventionby a general method using T4 DNA ligase and the like. For example, whenEscherichia coli is used, the vector can be introduced into a host bythe method of Hanahan et al. [Journal of Molecular Biology, 166, 557(1983)) and the like.

[0050] Also, the gene can also be isolated from a strain which isobtained by synthesizing an oligomer DNA based on the nucleotidesequence information of the G6PD gene (e.g., GenBank accession No.E13655 or the nucleotide sequence represented by SEQ ID NO:1 in the caseof Corynebacterium glutamicum), carrying out polymerase chain reaction(PCR) using the oligomer DNA as a primer and chromosomal DNA of amicroorganism belonging to the genus Corynebacterium as the template,ligating the obtained DNA fragment to a vector having a selection markergene and then introducing it into an appropriate host such as abacterium of the genus Escherichia or the genus Corynebacterium. In thiscase, it is not necessary to use a G6PD defective strain.

[0051] In addition, the gene can also be synthesized using a generallyused DNA synthesizer, such as ABI 3948 manufactured by Perkin-Elmer,based on a nucleotide sequence of the gene, for example, the nucleotidesequence represented by SEQ ID NO:1.

[0052] The DNA of the present invention isolated by the above method isintroduced into an expression vector which can replicate and express ina host microorganism, and the host microorganism is transformed with therecombinant vector thus obtained.

[0053] The recombinant DNA comprising the DNA encoding the polypeptideof the present invention is preferably a vector which can autonomouslyreplicate and which comprises a promoter, a ribosome binding sequence,the DNA of the present invention and a transcription terminationsequence. A gene for regulating the promoter may also be contained inthe recombinant DNA.

[0054] When a microorganism belonging to the genus Escherichia is used,examples of the vector for this object include pBTrp2, pBTac1 and pBTac2(all available from Boehringer Manheim), pKK233-2 (manufactured byPharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufacturedby Promega), pQE-8 (manufactured by QIAGEN), pKYP10 (Japanese PublishedUnexamined Patent Application No. 110600/83), pKYP200 [Agric. Biol.Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)],pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript IISK(−) (manufactured by Stratagene), pTrs30 [prepared from Escherichiacoil JM109/pTrS30 (FERM BP-5407)], pTrs32 [prepared from Escherichiacoli JM109/pTrS32 (FERM BP-5408)], pGHA2 [prepared, from Escherichiacoli IGHA2 (FERM B-400), Japanese Published Unexamined PatentApplication No. 221091/85], pGKA2 [prepared from Escherichia coil IGKA2(FERM BP-6798), Japanese Published Unexamined Patent Application No.221091/85], pTerm2 (U.S. Pat. No. 4,686,191, U.S. Pat. No. 4,939,094,U.S. Pat. No. 5,160,735), psupex, pUB110, pTP5, pC194, pEG400 [J.Bacteriol., 172, 2392 (1990)], pGEX (manufactured by Pharmacia) and pETsystem (manufactured by Novagen). When a microorganism belonging to thegenus Corynebacterium is used, examples include pCG1 (Japanese PublishedUnexamined Patent Application No. 134500/82), pCG2 (Japanese PublishedUnexamined Patent Application No. 35197/83), pCG4 (Japanese PublishedUnexamined Patent Application No. 183799/82), pCG11 (Japanese PublishedUnexamined Patent Application No. 134500/82), pCG116, pCES4 and pCB101(all Japanese Published Unexamined Patent Application No. 105999/83),pCE51, pCE52 and pCE53 [all Molecular and General Genetics, 196, 175(1984)] and pCS299P described in Examples of the present application.

[0055] Any promoter can be used, so long as it can function in the hostcell. Examples include promoters derived from Escherichia coli, phageand the like, such as trp promoter (P_(trp)), lac promoter, P_(L)promoter, P_(R) promoter, T7 promoter, and the like. Also, artificiallydesigned and modified promoters, such as a promoter in which two P_(trp)are linked in tandem (P_(trp)×2), tac promoter, lacT7 promoter, letIpromoter and the like, can be used.

[0056] It is preferred to use a plasmid in which the space betweenShine-Dalgarno sequence, which is the ribosome binding sequence, and theinitiation codon is adjusted to an appropriate distance (for example, 6to 18 bases).

[0057] In the recombinant DNA of the present invention, thetranscription termination sequence is not always necessary for theexpression of the DNA of the present invention. However, it is preferredto provide a transcription terminating sequence just downstream of thestructural gene.

[0058] Any host cell may be used, so long as it is a cell capable ofproducing L-amino acid described below. Preferably, a microorganismcapable of producing the amino acid is used. The microorganism is morepreferably a microorganism belonging to the genus Escherichia or thegenus Corynebacterium, still more preferably a microorganism belongingto the genus Corynebacterium, and most preferably Corynebacteriumglutamicum.

[0059] Examples of the microorganism include microorganisms belonging tothe genus Serratia, the genus Corynebacterium, the genus Arthrobacter,the genus Microbacterium, the genus Bacillus and the genus Escherichia.Specific examples include Escherichia coli XL1-Blue, Escherichia coliXL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichiacoli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichiacoli HB101, Escherichia coli No. 49, Escherichia coli W3110, Escherichiacoli NY49, Escherichia coli GI698, Escherichia coli TB1, Escherichiacoli ATCC 9637, Escherichia coli FERM BP-5985, Serratia ficaria,Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillussubtilis, Bacillus amyloliquefacines, Corynebacterium ammoniagenes ATCC6872, Brevibacterium immariophilium ATCC 14068, Brevibacteriumsaccharolyticum ATCC 14066, Brevibacterium roseum ATCC 13825,Brevibacterium thiogenitalis ATCC 19240, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC 13869, Corynebacterium glutamicumATCC 13032, Corynebacterium glutamicum ATCC 13869, Corynebacteriumglutamicum ATCC 13870, Corynebacterium callunae ATCC 15991,Corynebacterium acetoglutamicum ATCC 15806, Microbacterium ammoniaphilumATCC 15354 and Corynebacterium thermoaminogenes AJ 12340. The followingmicroorganism strain or a mutant strain producing L-amino acid derivedfrom the following microorganism strain is preferably used:

[0060]Corynebacterium glutamicum ATCC 13032;

[0061]Corynebacterium glutamicum ATCC 13869; and

[0062]Corynebacterium glutamicum ATCC 13870.

[0063] As the recombinant vector introducing-method, any method ofintroducing a DNA into the host cell can be used. For example, when amicroorganism belonging to the genus Escherichia is used, examplesinclude the method which comprises the use of a calcium ion [Proc. Natl.Acad. Sci. USA, 69, 2110 (1972)] and the electroporation method [Methodsin Enzymology, 235, 375 (1994)]. When a microorganism belonging to thegenus Corynebacterium is used, examples include the protoplast method(e.g., Japanese Published Unexamined Patent Application No. 186492/82and Japanese Published Unexamined Patent Application No. 18649/82), andthe electroporation method [e.g., Journal of Bacteriology, 175, 4096(1993)].

[0064] The microorganism belonging to the genus Escherichia or the genusCorynebacterium and comprising the DNA of the present invention on thechromosome may be any microorganism in which the DNA fragment isartificially integrated into the chromosome by a genetic recombinationor a mutagenizing treatment. For example, it may be a strain modified bya mutagenizing treatment from a strain containing a G6PD gene of anysequence into a strain comprising the DNA of the present invention, or astrain in which the DNA fragment is artificially integrated into thechromosome by the homologous recombination method [Bio/Technology, 9, 84(1991); Microbiology, 144, 1863 (1998)], the method which uses a phageor transposon [Escherichia coli and Salmonella typhimurium, 2325-2339(1996)] and the like. Preferably, a strain in which the DNA isintegrated into the chromosome by the homologous recombination method isexemplified.

[0065] In the present invention, a strain obtained by a mutagenizingtreatment as well as a strain obtained by a genetic recombination isalso called a transformant.

[0066] The polypeptide of the present invention can be produced byculturing the transformant of the present invention thus obtained in amedium to thereby form and accumulate the polypeptide of the presentinvention in the culture, and then recovering it from the culture.

[0067] Also, L-amino acid can be produced by culturing the transformantin a medium to thereby form and accumulate the L-amino acid in theculture, and then recovering it from the culture.

[0068] As the L-amino acid, any amino acid can be produced, so long asit needs NADUP for its biosynthesis. Examples include L-lysine,L-threonine, L-isoleucine, L-tryptophan, L-phenylalanine, L-tyrosine,L-histidine and L-cysteine. Also, a compound other than amino acidswhich uses these amino acids as intermediates can be produced.Preferably, L-lysine is exemplified. Biosynthetic pathways of aminoacids are shown in FIG. 1. In the drawing, reactions which consume NADPHare shown with an underline.

[0069] The transformant of the present invention can be cultured in amedium according to the usual method used for culturing a host.

[0070] As a medium used for culturing, the general nutritional mediumcontaining a carbon source, a nitrogen source, inorganic salts and thelike can be used.

[0071] Any carbon source which can be assimilated by the transformant orthe microorganism of the present invention is used. Examples includecarbohydrates such as glucose, fructose, sucrose, molasses containingthem, starch, starch hydrolysate, etc.; organic acids such as aceticacid, propionic acid, etc.; and alcohols such as ethanol, propanol, etc.

[0072] Examples of the nitrogen source include ammonia; ammonium saltsof inorganic acids or organic acids such as ammonium chloride, ammoniumsulfate, ammonium acetate, ammonium phosphate, etc.; othernitrogen-containing compounds; peptone; meat extract; yeast extract;corn steep liquor; casein hydrolysate; soybean meal and soybean mealhydrolysate; and various cells obtained by fermentation and theirdigested products.

[0073] Examples of the inorganic salts include potassium dihydrogenphosphate, dipotassium : hydrogen phosphate, magnesium phosphate,magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate,copper sulfate and calcium carbonate.

[0074] Culturing is carried out under aerobic conditions by shakingculture, submerged spinner culture under aeration or the like. Theculturing temperature is preferably from 15 to 40° C., and the culturingtime is generally from 16 hours to 7 days. The pH during the culturingis preferably maintained at 3.0 to 9.0. The pH can be adjusted using aninorganic or organic acid, an alkali solution, urea, calcium carbonate,ammonia or the like.

[0075] Also, antibiotics such as ampicillin, tetracycline, and the likecan be added to the medium during culturing, if necessary.

[0076] When a microorganism transformed with a recombinant vectorharboring an inducible promoter as the promoter is cultured, an inducermay be added to the medium, if necessary. For example, when amicroorganism transformed with a recombinant vector harboring lacpromoter is cultured, isopropyl-β-D-thiogalactopyranoside (IPTG) or thelike may be added to the medium, or when a microorganism transformedwith a recombinant vector harboring trp promoter is cultured,indoleacrylic acid or the like may be added to the medium.

[0077] After culturing, precipitates such as cells and the like areremoved from the culture, and L-amino acid can be recovered from theculture using ion exchange treatment, concentration, salting out and thelike in combination.

[0078] The polypeptide produced by the transformant of the presentinvention can be isolated and purified using the usual method forisolating and purifying an enzyme. For example, when the polypeptide ofthe present invention is expressed as a soluble product in the hostcells, the cells are recovered by centrifugation after culturing,suspended in an aqueous buffer, and are disrupted using analtrasonicator, a French press, a Manton Gaulin homogenizer, a Dynomillor the like to obtain a cell-free extract solution. From the supernatantobtained by centrifuging the cell-free extract solution, a purifiedproduct can be obtained by the usual method used for isolating andpurifying an enzyme, for example, solvent extraction; salting out usingammonium sulfate or the like; desalting; precipitation using an organicsolvent; anion exchange chromatography using a resin, such asdiethylaminoethyl (DEAE)-Sepharose, DIAION HPA-75 (manufactured byMitsubishi Chemical) etc.; cation exchange chromatography using a resin,such as S-Sepharose FF (manufactured by Pharmacia) etc.; hydrophobicchromatography using a resin, such as butyl sepharose, phenyl sepharoseetc.; gel filtration using a molecular sieve; affinity chromatography,chromatofocusing; electrophoresis, such as isoelectronic focusing etc.;and the like alone or in combination thereof.

[0079] When the polypeptide is expressed as an inclusion bodyintracellularly, the cells are recovered in the same manner, disruptedand centrifuged to recover the polypeptide as the precipitate fraction.The inclusion body of the recovered polypeptide is solubilized with aprotein denaturing agent. The solubilized polypeptide solution isdiluted or dialyzed to lower the concentration of the protein denaturingagent in the solution to thereby restore the normal tertiary structureof the polypeptide. After the procedure, a purified product of thepolypeptide can be obtained by a purification/isolation method similarto the above.

[0080] When the polypeptide of the present invention is secretedextracellularly, the polypeptide can be recovered in the culturesupernatant. Specifically, the culture supernatant is obtained bytreating the culture in a treatment similar to the above, such ascentrifugation or the like. Then, a purified product can be obtainedfrom the supernatant using a purification/isolation method similar tothe above.

[0081] Examples of the polypeptide thus obtained include a polypeptidecomprising the amino acid sequence represented by SEQ ID NO:2 or 12.

[0082] Also, the polypeptide of the present invention can be produced bya chemical synthesis method, such as Fmoc (fluorenylmethyloxycarbonyl)method, tBoc (t-butyloxycarbonyl) method or the like. Furthermore, itcan be chemically synthesized using a peptide synthesizer manufacturedby Advanced ChemTech, Perkin-Elmer, Pharmacia, Protein TechnologyInstrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation or thelike.

[0083] Examples of the present invention are shown below; however, thepresent invention is not limited to these Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

[0084]FIG. 1 shows biosynthetic pathways of 20 amino acids constitutingproteins in a bacterium of the genus Corynebacterium. The underlinedparts show reactions which consume NADPH. The framed parts showreactions which produce NADPH.

[0085] The genes which correspond to enzymes relating to respectivereactions are named basically by the nomenclature of Escherichia coil.In the drawing, glucose-6-phosphate dehydrogenase is represented byG6PD(zwf).

[0086]FIG. 2 shows construction steps of pCS299P.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1

[0087] Preparation of Novel G6PD Gene:

[0088] (1) Determination of G6PD Gene Nucleotide Sequence

[0089]Corynebacterium glutamicum No. 58 (hereinafter referred to as “No.58 strain”) is an L-lysine producing strain obtained by applying amutagenizing operation to Corynebacterium glutamicum ATCC 13032(hereinafter referred to as “ATCC 13032 strain”).

[0090] The strain has been deposited on Apr. 14, 2000, in InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology, AIST Tsukuba Central 6, 1-1, Higashi 1-ChomeTsukuba-shi, Ibaraki-ken, Japan (the old name: National Institute ofBioscience and Human technology, Agency of Industrial Science andTechnology: 1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan) withaccession number FERM BP-7134. G6PD genes of the ATCC 13032 strain andNo. 58 strain were cloned as follows.

[0091] A chromosomal DNA was prepared from each strain by the method ofSaito et al. [Biochimica et Biophysica Acta, 72, 619 (1963)]. Also,based on the G6PD gene nucleotide sequence already known inCorynebacterium glutamicum MJ233 (GenBank accession number E13655), PCRprimers for the target nucleotide sequence were prepared by the usualmethod. Nucleotide sequences of the primers are shown in SEQ ID NOs:3and 4. PCR was carried out by 25 cycles, one cycle consisting of areaction at 94° C. for 1 minute, reaction at 60° C. for 1 minute andreaction at 74° C. for 2 minutes, using a thermal cycler manufactured byPerkin-Elmer (GeneAmp PCR System 9600), Pfu turbo DNA polymerase(manufactured by Stratagene), 100 ng of each chromosomal DNA and theattached-buffer. An amplified PCR product of about 2.2 kb was subjectedto agarose gel electrophoresis and extracted and purified using QIAquickGel Extraction Kit (manufactured by Quiagen).

[0092] The above 2.2 kb DNA fragment containing the G6PD gene and apCR-Blunt vector (manufactured by Invitrogen) were ligated using T4 DNAligase (manufactured by Takara Shuzo), which was used to transformEscherichia coli One Shot TOP10 competent cells (manufactured byInvitrogen) according to the usual method. Each of the transformantsselected on an LB agar medium [medium containing 5 g of Yeast Extract(manufactured by Difco), 10 g of Bacto-tryptone (manufactured by Difco),10 g of sodium chloride and 16 g of agar (manufactured by Ditco) in 1liter of water and adjusted to pH 7.2] containing 50 μg/ml kanamycin wascultured overnight in LB medium containing 50 μg/ml kanamycin, andplasmids were prepared from the respective culture media thus obtainedby the alkaline SDS method (Molecular Cloning, Second Edition).

[0093] A plasmid containing the G6PD gene derived from the ATCC 13032strain was named pCRBzwf1, and a plasmid containing the G6PD genederived from the No. 58 strain was named pCRBzwf2.

[0094] Next, nucleotide sequences of G6PD gene on the plasmids weredetermined by the conventional method. AS a result, it was found thatthe nucleotide sequences of G6PD genes obtained from the ATCC 13032strain and the No. 58 strain were completely the same. The nucleotidesequence is shown in SEQ ID NO:1. That is, it was shown that the G6PDgene of the L-lysine producing strain No. 58 is a wild-type one.

[0095] (2) Preparation of Novel G6PD Gene

[0096] No. 58 strain was subjected to a mutagenizing treatment with NTG(Microbial Experimentation Manual, 1986, p. 131, Kodansha Scientific)and then inoculated onto a minimal agar medium [a medium containing 10 gof glucose, 4 g of ammonium chloride, 2 g of urea, 1 g of potassiumdihydrogenphosphate, 3 g of dipotassium hydrogenphosphate, 4 mg offerrous sulfate heptahydrate, 40 μg of zinc chloride heptahydrate, 200μg of ferric chloride hexahydrate, 10 μg of copper chloride dihydrate,10 μg of manganese chloride tetrahydrate, 10 μg of sodium tetraboratedecahydrate, 10 μg of ammonium molybdate tetrahydrate, 50 μg of biotin,5 mg of nicotinic acid and 16 g of agar (manufactured by Difco) in 1liter of water and adjusted to pH 7.2] containing 1 mg/ml 6-azauraciland cultured at 30° C. for 2 days. The thus formed colonies wereisolated and subjected to the L-lysine production test described inExample 2(4) below, clones having higher productivity than that of No.58 strain were selected. Among these, one strain was named Ml strain.G6PD gene of M1 strain was isolated by the method of (1), and the genewas inserted into the pCR-Blunt vector. The thus obtained recombinantplasmid was named pCRBzwfM. When its nucleotide sequence was determined,the nucleotide at position 637 of SEQ ID NO:1, which is guanine in theG6PD genes of the ATCC 13032 strain and No. 58 strain, was changed toadenine in the G6PD gene of M1 strain. The nucleotide sequence is shownin SEQ ID NO:11.

[0097] As a result of the mutation, Ala at position 213 (codon GCT) fromthe amino terminal side of the G6PD in the ATCC 13032 strain and No. 58strain was changed to Thr (codon ACT) in the G6PD in the M1 strain. Theamino acid sequence was shown in SEQ ID NO;12.

[0098] That is, it was shown that an amino acid substitution mutation ofAla213Thr is present in the G6PD of the M1 strain. Escherichia coliTOP10 comprising the pCRBzwfM has been deposited on Apr. 14, 2000, inInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology, AIST Tsukuba Central 6, 1-1, Higashi1-Chome Tsukuba-shi, Ibaraki-ken, Japan (the old name: NationalInstitute of Bioscience and Human technology, Agency of IndustrialScience and Technology; 1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken,Japan) with accession number FERM BP-7135.

EXAMPLE 2

[0099] Effect of the Novel G6PD Gene L-Lysine Production:

[0100] (1) Construction of Vector for Gene Substitution

[0101] In order to examine the effect of amino acid substitutionmutation in G6PD shown in Example 1, the G6PD gene of No. 58 strain wassubstituted into a mutant.

[0102] A vector for gene substitution for this substitution wasconstructed as follows.

[0103] Single-stranded 37 mer DNA of and 29 mer DNA comprising thenucleotide sequences represented by SEQ ID NOs:5 and 6, respectively,were synthesized according to the conventional method. They were mixedin 50 μl of 0.1 M NaCl to give a respective concentration of 10pmole/μl, and allowed to stand at 95° C. for 2 minutes and then at 65°C. for 15 minutes. After cooling to 30° C. over 3 hours, both of thesingle-stranded DNA molecules were paired to obtain a double-strandedDNA.

[0104] pHSG299 (manufactured by Takara Shuzo) was digested with EcoRIand SphI (both manufactured by Takara Shuzo), subjected to agarose gelelectrophoresis and then extracted and purified using QIAquick GelExtraction Kit (manufactured by Quiagen). The thus obtained pHSG299fragment was ligated with the above double-stranded DNA fragment byusing Ligation Kit ver. 2 (manufactured by Takara Shuzo), andEscherichia coli DH5α was transformed therewith according to theconventional method. The strain was cultured on LB agar mediumcontaining 50 μg/ml kanamycin, and transformants were selected. Amongthe transformants, one strain was cultured overnight in LB mediumcontaining 50 μg/ml kanamycin, and a plasmid was prepared from theculture medium thus obtained by the alkaline SDS method. The thusobtained plasmid was named pHSG299L.

[0105] (2) Construction of Plasmid pCS299P

[0106] A shuttle vector pCS299P which is autonomously replicable in bothEscherichia coli and a coryneform bacterium was prepared by thefollowing method.

[0107] A BglII-digested fragment was obtained by digesting pCG116[Bio/Technology, 11, 921 (1993)] with BglII (manufactured by TakaraShuzo).

[0108] After digesting pHSG299 (manufactured by Takara Shuzo) with BamHI(manufactured by Takara Shuzo), the thus obtained BamHI-digestedfragment was concentrated by ethanol precipitation according to theconventional method, and the fragment was treated with alkalinephosphatase. The two fragments thus obtained were mixed and subjected toa ligation reaction by using Ligation Kit. ver. 1 (manufactured byTakara Shuzo). Using the reaction product, Escherichia coli NM522 wastransformed according to the conventional method (Molecular Cloning,Second Edition). The strain was cultured on LB agar medium containing 20μg/ml kanamycin to select a transformant. The transformant was culturedovernight in LB agar medium containing 20 μg/ml kanamycin, and a plasmidwas prepared from the culture thus obtained by the alkaline SDS methodto obtain pCS116-299Bgl1 DNA.

[0109] Restriction sites of the pCS116-299Bgl1 DNA were confirmedaccording to the conventional method.

[0110] Using the pCS116-299Bgl1 DNA, Corynebacterium ammoniagenes ATCC6872 was transformed by electroporation [FEMS Microbiology Letters, 65,299 (1989)).

[0111] A transformant was selected by culturing the strain on a CM agarmedium [a medium containing 10 g of Polypeptone S (manufactured by NihonPharmaceutical), 5 g of Yeast extract S (manufactured by NihonPharmaceutical), 10 g of Ehrlich meat extract (manufactured by KyokutoPharmaceutical), 3 g of sodium chloride and 30 μg of biotin in 1 literof water and adjusted to pH 7.2] containing 20 μg/ml kanamycin. Aplasmid was extracted from the transformant according to theconventional method, and the plasmid was digested with restrictionenzymes to confirm that the plasmid is pCS116-299Bgl1.

[0112] The pCS116-299Bgl1 DNA was digested with PstI (manufactured byTakara Shuzo) and BamHI and then purified by ethanol precipitation. Apartially deleted plasmid was prepared from the thus obtained DNA usinga deletion kit for kilo-sequencing (manufactured by Takara Shuzo).Escherichia coli NM522 was transformed using the plasmid according tothe conventional method. The strain was cultured on LB agar mediumcontaining 20 μg/ml kanamycin to select transformants. The transformantswere cultured overnight in LB medium containing 20 μg/ml kanamycin, andplasmids were prepared from the culture medium thus obtained by thealkaline SDS method. According to the conventional method, a restrictionmap of each of the thus obtained plasmids was prepared, and plasmidshaving a different partially-deleted length were selected.

[0113]Corynebacterium ammoniagenes ATCC 6872 was transformed using theplasmids by electroporation thus selected. The transformants thusobtained were spread on CM agar medium containing 20 μg/ml kanamycin andcultured at 30° C. for 2 days, and plasmids which was autonomouslyreplicable in Corynebacterium ammoniagenes were selected based onwhether kanamycin resistant colonies were formed or not.

[0114] Among the plasmids having autonomous replication ability, aplasmid having the longest deletion region was selected, and thisplasmid was named pCS299de16.

[0115] The pCS299de16 DNA was prepared from the transformant accordingto the conventional method and then digested with restriction enzymesDraI and PvuII (both manufactured by Takara Shuzo). The digested DNAfragments were fractionated by agarose gel electrophoresis, and about2.7 kb DNA fragment having a pCG116-derived DNA was separated and thenextracted and purified by using DNA prep (manufactured by Asahi Glass).

[0116] The DNA of pBluescript SK(+) (manufactured by TOYOBO) wasdigested with EcoRV (manufactured by Takara Shuzo) according to theconventional method. The thus digested DNA fragments were concentratedby ethanol precipitation and then subjected to alkaline phosphatasetreatment. The treated DNA fragments were fractionated by agarose gelelectrophoresis and then extracted and purified using the DNA prep.

[0117] The 2.7 kb DNA fragment and pBluescript SK(+) fragment wereligated using the Ligation Kit ver. 1, and then the Escherichia coliNM522 was transformed by using the ligated DNA according to theconventional method. The strain was cultured on LB agar mediumcontaining 100 μg/ml ampicillin, 50 μg/ml X-Gal(5-bromo-4-chloro-3-indoyl-β-D-galactoside) and 1 mmol/l IPTG(isopropylthio-β-D-galactoside) to select transformants. Thetransformants were cultured overnight in LB medium containing 100 μg/mlampicillin, and plasmids were prepared from the culture thus obtained bythe alkaline SDS method. According to the conventional method, arestriction map of each of the thus obtained plasmids was prepared. Aplasmid capable of forming 3.4 kb DNA fragment and 2 kb DNA fragment byEcoRI digestion was named pCSSK21.

[0118] DNA fragments having the nucleotide sequences represented by SEQID NOs;7 and 8 were synthesized, and PCR was carried out by using theDNA fragments as primers, and the pHSG299 DNA as the template, and usingTaq DNA polymerase (manufactured by Takara Shuzo) according to thereaction conditions attached thereto. The reaction product wasprecipitated with ethanol according to the conventional method and thendigested with restriction enzymes PstI and XhoI (manufactured by TakaraShuzo). The digested DNA fragments were fractionated by agarose gelelectrophoresis, and the about 1.3 kb DNA fragment thus obtained wasextracted and purified using the DNA prep.

[0119] DNA fragments having the nucleotide sequences represented by SEQID NOs:9 and 10 were synthesized, and PCR was carried out by using theTaq DNA polymerase according to the reaction conditions attachedthereto, wherein the DNA fragments were used as primers, and the pHSG299DNA was used as the template. The reaction product was precipitated withethanol according to the conventional method and then digested withrestriction enzymes PstI and BglII. The digested DNA fragments werefractionated by agarose gel electrophoresis, and the about 1.3 kb DNAfragment thus obtained was extracted and purified using the DNA prep.

[0120] The plasmid pCSSK21 thus obtained was digested with SalI(manufactured by Takara Shuzo) and BamHI. The digested DNA fragmentswere fractionated by agarose gel electrophoresis, and the about 2.7 kbDNA fragment thus obtained was extracted and purified by using the DNAprep. The three DNA fragments extracted and purified above were mixedand then ligated by using the Ligation Kit ver. 1.

[0121] The Escherichia coli NM522 was transformed with the ligated DNAfragment according to the conventional method. The strain was culturedon LB agar medium containing 20 μg/ml kanamycin, 50 μg/ml X-Gal and 1mmol/l IPTG to select transformants.

[0122] The transformants were cultured overnight in LB medium containing20 μg/ml kanamycin, and plasmids were prepared from the culture mediumthus obtained by the alkaline SDS method. According to the conventionalmethod, a restriction map of each of the thus obtained plasmids wasprepared, and the plasmid having the structure described in FIG. 1 wasnamed pCS299P.

[0123] The plasmids pCS299P and pHSG299L were digested with XbaI andPstI (both manufactured by Takara Shuzo) and then subjected to agarosegel electrophoresis. Each of the 2.5 kb fragment containing apCS299P-derived replication initiation region (Oric) in bacteria of thegenus Corynebacterium and the pHSG299L fragment was extracted andpurified by using QIAquick Gel Extraction Kit (manufactured by QUIAGEN).The 2.5 kb DNA fragment and the pHSG299L fragment were ligated by usingLigation Kit ver. 2 (manufactured by Takara Shuzo) and used to transforminto Escherichia coli DHα according to the conventional method. Aplasmid was prepared from the thus obtained transformant in the samemanner as the method. The thus obtained plasmid was named pHSG2990C.

[0124] Plasmids pMOB3 (ATCC 77282) and pHSG299OC were digested with PstI(manufactured by Takara Shuzo) and then subjected to agarose gelelectrophoresis. Each of the 2.6 kb fragment containing a pMOB3-derivedBacillus subtilis levan sucrase (SacB) gene and the pHSG299OC fragmentwas extracted and purified by using QIAquick Gel Extraction Kit(manufactured by QUIAGEN).

[0125] The 2.6 kb DNA fragment and the pHSG299OC fragment were ligatedby using Ligation Kit ver. 2 (manufactured by Takara Shuzo) andtransformed into Escherichia coli DHα according to the conventionalmethod. The strain was cultured on LB agar medium containing 50 μg/mlkanamycin to select a transformant. A plasmid was prepared from the thusobtained transformant in the same manner as the above method. Theplasmid was named pHSG299OCSB.

[0126] A 5.1 kb DNA fragment obtained by digesting the pHSG299OCSB withNotI was subjected to agarose gel electrophoresis and then extracted andpurified by using QIAquick Gel Extraction Kit (manufactured by QUIAGEN).pCRBzwfM prepared in Example 1 was digested with NotI, subjected toagarose gel electrophoresis and then extracted and purified by usingQIAquick Gel Extraction Kit (manufactured by QUIAGEN). A NotI fragmentcontaining Oric and SacB gene was connected to the NotI site of pCRBzwfMby using Ligation Kit ver. 2 (manufactured by Takara Shuzo) andtransformed into Escherichia coli DH5α according to the conventionalmethod. The strain was cultured on LB agar medium containing 50 μg/mlkanamycin to select a transformant. A plasmid was prepared from the thusobtained transformant in the same manner as the above method. Theplasmid was named pCRBOSzwfM and used as a recombinant vector for G6PDgene.

[0127] (3) Substitution of G6PD Gene of No. 58 Strain

[0128] The pCRBOSzwfM containing mutant G6PD gene was introduced intothe No. 58 strain and then integrated into chromosomal DNA by homologousrecombination using the method of Ikeda et al. [Microbiology, 144, 1863(1998)].

[0129] Strains in which second homologous recombination was occurredwere selected by the selection method which uses a property of theBacillus subtilis levan sucrase encoded by pCRBOSzwfM to produce asuicide substrate [Journal of Bacteriology, 174, 5462 (1992)], and astrain in which the G6PD gene (wild-type) originally contained in theNo. 58 strain was substituted with the mutant G6PD gene was isolatedfrom the above selected strains by the following method.

[0130] The pCRBOSzwfM was introduced into the No. 58 strain byelectroporation [FEMS Microbiology Letters, 65, 299 (1989)], andtransformants were obtained by culturing the strain at 30° C. for 2hours on KM163 agar medium [a medium containing 10 g of glucose, 10 g ofPeptone (manufactured by Kyokuto Pharmaceutical), 5 g of Ehrlich meatextract (manufactured by Kyokuto Pharmaceutical), 2 g of urea, 2.5 g ofsodium chloride and 18 g of Bacto-agar (manufactured by Difco) in 1liter of water and adjusted to pH 7.23 containing 50 μg/ml kanamycin. Astrain Tf1 as one of the transformants was selected, and the strain wascultured in KM163 medium containing 20 μg/ml kanamycin and subjected toelectroporation to introduce pCGll (Japanese Patent Publication No.91827/1994). After the introduction operation, the strain was culturedon KM163 agar medium containing 50 μg/ml kanamycin and 200 μg/mlspectinomycin at 30° C. for 2 days to obtain transformants. Chromosomeof a strain from the transformants was examined by Southern blothybridization according to the method of Ikeda et al. [Microbiology,144, 1863 (1998)]. As a result, it was confirmed that the pCRBOSzwfM wasintegrated into the chromosome by a Campbell-type homologousrecombination. Since the wild-type and mutant G6PD genes are closelylocated on the chromosome in those strains, second homologousrecombination is apt to occur between them.

[0131] The transformant (single recombinant) was spread on a Suc medium[a medium containing 100 g of sucrose, 7 g of meat extract, 10 g ofpeptone, 3 g of sodium chloride, 5 g of Yeast extract (manufactured byDifco) and 18 g of Bacto-agar (manufactured by Difco) in 1 liter ofwater and adjusted to pH 7.2] and cultured at 30° C. for 1 day, and thesurviving colonies were selected. A strain having the SacB gene cannotgrow on this medium because it converts sucrose into a suicidesubstrate. On the other hand, a strain in which the SacB gene is deletedby the second homologous recombination between the wild-type and mutantG6PD genes can grow on this medium because the suicide substrate is notformed. During the homologous recombination, either the wild-type ormutant G6PD gene is deleted together with SacB. In this case, genesubstitution into the mutant G6PD gene occurs in a strain in which thewild-type G6PD gene is deleted together with SacB.

[0132] A chromosomal DNA of the secondary recombinant obtained above wasprepared by the method of Saito et al. [Biochimica et Biophysica Acta,12, 619 (1963)], and PCR was carried out by using Pfu turbo DNApolymerase (manufactured by Stratagene) and the buffer attached thereto,wherein DNA fragments having the nucleotide sequences represented by SEQID NOs:3 and 4 were used as primers. Typing of the G6PD gene of thedouble recombinant in terms of wild-type or mutant was done bydetermining the nucleotide sequences of these PCR products in the usualway. As the results, it was confirmed that strains having only thewild-type G6PD gene (No. 58W strain as an example) and strains havingonly the mutant G6PD gene (No. 58M strain as an example) were obtained.

[0133] (4) L-Lysine Production Test

[0134] Lysine productivity of the thus obtained G6PD gene-substitutedstrains (No. 58W and No. 58M) and the No. 58 strain as the parent strainwas evaluated by culturing them using a 5 liter-jar fermentor.

[0135] Each strain was inoculated into 100 ml of a first seed medium [amedium prepared by dissolving 50 g of glucose, 10 g of Yeast extract(manufactured by Nihon Pharmaceutical), 10 g of Peptone (manufactured byKyokuto Pharmaceutical Industry), 5 g of corn steep liquor, 2.5 g ofsodium chloride, 3 g of urea and 50 μg of biotin in 1 liter of water,adjusting the solution to pH 7.2, and further adding 10 g of calciumcarbonate), and cultured at 30° C. for 24 hours in a 1 liter capacityErlenmeyer flask with baffles. Next, 40 ml of the first seed broth wasinoculated into 2,000 ml of a second seed medium (a medium prepared bydissolving 50 g of glucose, 10 g of corn steep liquor, 0.5 g ofmagnesium sulfate heptahydrate, 5 mg of nicotinic acid, 1 mg of thiaminhydrochloride, 100 μg of biotin, 10 mg of calcium pantothenate, 2 g ofpotassium dihydrogenphosphate, 3 g of urea, 10 mg of ferrous sulfateheptahydrate, 1 mg of zinc sulfate heptahydrate, 8 g of ammoniumsulfate, 20 g of peptone and 2 g of sodium bicarbonate in I liter ofwater), and cultured at 30° C. for 12 hours in a 5 liter-jar fermentor.Next, 230 ml of the second seed broth was inoculated into 1,675 ml of amain culture medium [a medium prepared by dissolving 93 g of blackstrapmolasses (sugar equivalent amount), 0.5 g of potassiumdihydrogenphosphate, 10 mg of ferrous sulfate heptahydrate, 100 μg ofthiamin hydrochloride, 2 g of soy peptone, 0.5 g of magnesium sulfateheptahydrate, 5 mg of nicotinic acid and 15 g of ammonium sulfate in 1liter of water, and adjusted the pH to 7.4], and cultured at 35° C. for42 hours in a 5 liter-jar fermentor.

[0136] The amount of L-lysine accumulated in the main culture wasquantified by high performance liquid chromatography (HPLC).

[0137] Table 1 shows results of the measurement of the amount ofL-lysine produced by the No. 5.8 strain, No. 58W strain and No. 58Mstrain. The results show that the L-lysine productivity is improved bythe novel mutant C6PD. TABLE 1 Strain L-Lysine productivity (g/l) No. 5849.7 No. 58W 53.5 No. 58M 63.3

[0138] Industrial Applicability

[0139] According to the present invention, a modified G6PD and a DNAencoding the G6PD are obtained, and the productivity of L-amino acid bya microorganism can be improved by using the modified G6PD.

[0140] Free Text of Sequence Listing:

[0141] SEQ ID NO:3: Description of artificial sequence—Synthetic DNA

[0142] SEQ ID NO:4: Description of artificial sequence—Synthetic DNA

[0143] SEQ ID NO:5: Description of artificial sequence—Synthetic DNA

[0144] SEQ ID NO:6: Description of artificial sequence—Synthetic DNA

[0145] SEQ ID NO:7: Description of artificial sequence—Synthetic DNA

[0146] SEQ ID NO:8: Description of artificial sequence—Synthetic DNA

[0147] SEQ ID NO:9: Description of artificial sequence—Synthetic DNA

[0148] SEQ ID NO:10: Description of artificial sequence—Synthetic DNA

1 12 1 1452 DNA Corynebacterium glutamicum CDS (1)..(1452) 1 atg gtg atcttc ggt gtc act ggc gac ttg gct cga aag aag ctg ctc 48 Met Val Ile PheGly Val Thr Gly Asp Leu Ala Arg Lys Lys Leu Leu 1 5 10 15 ccc gcc atttat gat cta gca aac cgc gga ttg ctg ccc cca gga ttc 96 Pro Ala Ile TyrAsp Leu Ala Asn Arg Gly Leu Leu Pro Pro Gly Phe 20 25 30 tcg ttg gta ggttac ggc cgc cgc gaa tgg tcc aaa gaa gac ttt gaa 144 Ser Leu Val Gly TyrGly Arg Arg Glu Trp Ser Lys Glu Asp Phe Glu 35 40 45 aaa tac gta cgc gatgcc gca agt gct ggt gct cgt acg gaa ttc cgt 192 Lys Tyr Val Arg Asp AlaAla Ser Ala Gly Ala Arg Thr Glu Phe Arg 50 55 60 gaa aat gtt tgg gag cgcctc gcc gag ggt atg gaa ttt gtt cgc ggc 240 Glu Asn Val Trp Glu Arg LeuAla Glu Gly Met Glu Phe Val Arg Gly 65 70 75 80 aac ttt gat gat gat gcagct ttc gac aac ctc gct gca aca ctc aag 288 Asn Phe Asp Asp Asp Ala AlaPhe Asp Asn Leu Ala Ala Thr Leu Lys 85 90 95 cgc atc gac aaa acc cgc ggcacc gcc ggc aac tgg gct tac tac ctg 336 Arg Ile Asp Lys Thr Arg Gly ThrAla Gly Asn Trp Ala Tyr Tyr Leu 100 105 110 tcc att cca cca gat tcc ttcaca gcg gtc tgc cac cag ctg gag cgt 384 Ser Ile Pro Pro Asp Ser Phe ThrAla Val Cys His Gln Leu Glu Arg 115 120 125 tcc ggc atg gct gaa tcc accgaa gaa gca tgg cgc cgc gtg atc atc 432 Ser Gly Met Ala Glu Ser Thr GluGlu Ala Trp Arg Arg Val Ile Ile 130 135 140 gag aag cct ttc ggc cac aacctc gaa tcc gca cac gag ctc aac cag 480 Glu Lys Pro Phe Gly His Asn LeuGlu Ser Ala His Glu Leu Asn Gln 145 150 155 160 ctg gtc aac gca gtc ttccca gaa tct tct gtg ttc cgc atc gac cac 528 Leu Val Asn Ala Val Phe ProGlu Ser Ser Val Phe Arg Ile Asp His 165 170 175 tat ttg ggc aag gaa acagtt caa aac atc ctg gct ctg cgt ttt gct 576 Tyr Leu Gly Lys Glu Thr ValGln Asn Ile Leu Ala Leu Arg Phe Ala 180 185 190 aac cag ctg ttt gag ccactg tgg aac tcc aac tac gtt gac cac gtc 624 Asn Gln Leu Phe Glu Pro LeuTrp Asn Ser Asn Tyr Val Asp His Val 195 200 205 cag atc acc atg gct gaagat att ggc ttg ggt gga cgt gct ggt tac 672 Gln Ile Thr Met Ala Glu AspIle Gly Leu Gly Gly Arg Ala Gly Tyr 210 215 220 tac gac ggc atc ggc gcagcc cgc gac gtc atc cag aac cac ctg atc 720 Tyr Asp Gly Ile Gly Ala AlaArg Asp Val Ile Gln Asn His Leu Ile 225 230 235 240 cag ctc ttg gct ctggtt gcc atg gaa gaa cca att tct ttc gtg cca 768 Gln Leu Leu Ala Leu ValAla Met Glu Glu Pro Ile Ser Phe Val Pro 245 250 255 gcg cag ctg cag gcagaa aag atc aag gtg ctc tct gcg aca aag ccg 816 Ala Gln Leu Gln Ala GluLys Ile Lys Val Leu Ser Ala Thr Lys Pro 260 265 270 tgc tac cca ttg gataaa acc tcc gct cgt ggt cag tac gct gcc ggt 864 Cys Tyr Pro Leu Asp LysThr Ser Ala Arg Gly Gln Tyr Ala Ala Gly 275 280 285 tgg cag ggc tct gagtta gtc aag gga ctt cgc gaa gaa gat ggc ttc 912 Trp Gln Gly Ser Glu LeuVal Lys Gly Leu Arg Glu Glu Asp Gly Phe 290 295 300 aac cct gag tcc accact gag act ttt gcg gct tgt acc tta gag atc 960 Asn Pro Glu Ser Thr ThrGlu Thr Phe Ala Ala Cys Thr Leu Glu Ile 305 310 315 320 acg tct cgt cgctgg gct ggt gtg ccg ttc tac ctg cgc acc ggt aag 1008 Thr Ser Arg Arg TrpAla Gly Val Pro Phe Tyr Leu Arg Thr Gly Lys 325 330 335 cgt ctt ggt cgccgt gtt act gag att gcc gtg gtg ttt aaa gac gca 1056 Arg Leu Gly Arg ArgVal Thr Glu Ile Ala Val Val Phe Lys Asp Ala 340 345 350 cca cac cag cctttc gac ggc gac atg act gta tcc ctt ggc caa aac 1104 Pro His Gln Pro PheAsp Gly Asp Met Thr Val Ser Leu Gly Gln Asn 355 360 365 gcc atc gtg attcgc gtg cag cct gat gaa ggt gtg ctc atc cgc ttc 1152 Ala Ile Val Ile ArgVal Gln Pro Asp Glu Gly Val Leu Ile Arg Phe 370 375 380 ggt tcc aag gttcca ggt tct gcc atg gaa gtc cgt gac gtc aac atg 1200 Gly Ser Lys Val ProGly Ser Ala Met Glu Val Arg Asp Val Asn Met 385 390 395 400 gac ttc tcctac tca gaa tcc ttc act gaa gaa tca cct gaa gca tac 1248 Asp Phe Ser TyrSer Glu Ser Phe Thr Glu Glu Ser Pro Glu Ala Tyr 405 410 415 gag cgc ctcatt ttg gat gcg ctg tta gat gaa tcc agc ctc ttc cct 1296 Glu Arg Leu IleLeu Asp Ala Leu Leu Asp Glu Ser Ser Leu Phe Pro 420 425 430 acc aac gaggaa gtg gaa ctg agc tgg aag att ctg gat cca att ctt 1344 Thr Asn Glu GluVal Glu Leu Ser Trp Lys Ile Leu Asp Pro Ile Leu 435 440 445 gaa gca tgggat gcc gat gga gaa cca gag gat tac cca gcg ggt acg 1392 Glu Ala Trp AspAla Asp Gly Glu Pro Glu Asp Tyr Pro Ala Gly Thr 450 455 460 tgg ggt ccaaag agc gct gat gaa atg ctt tcc cgc aac ggt cac acc 1440 Trp Gly Pro LysSer Ala Asp Glu Met Leu Ser Arg Asn Gly His Thr 465 470 475 480 tgg cgcagg cca 1452 Trp Arg Arg Pro 2 484 PRT Corynebacterium glutamicum 2 MetVal Ile Phe Gly Val Thr Gly Asp Leu Ala Arg Lys Lys Leu Leu 1 5 10 15Pro Ala Ile Tyr Asp Leu Ala Asn Arg Gly Leu Leu Pro Pro Gly Phe 20 25 30Ser Leu Val Gly Tyr Gly Arg Arg Glu Trp Ser Lys Glu Asp Phe Glu 35 40 45Lys Tyr Val Arg Asp Ala Ala Ser Ala Gly Ala Arg Thr Glu Phe Arg 50 55 60Glu Asn Val Trp Glu Arg Leu Ala Glu Gly Met Glu Phe Val Arg Gly 65 70 7580 Asn Phe Asp Asp Asp Ala Ala Phe Asp Asn Leu Ala Ala Thr Leu Lys 85 9095 Arg Ile Asp Lys Thr Arg Gly Thr Ala Gly Asn Trp Ala Tyr Tyr Leu 100105 110 Ser Ile Pro Pro Asp Ser Phe Thr Ala Val Cys His Gln Leu Glu Arg115 120 125 Ser Gly Met Ala Glu Ser Thr Glu Glu Ala Trp Arg Arg Val IleIle 130 135 140 Glu Lys Pro Phe Gly His Asn Leu Glu Ser Ala His Glu LeuAsn Gln 145 150 155 160 Leu Val Asn Ala Val Phe Pro Glu Ser Ser Val PheArg Ile Asp His 165 170 175 Tyr Leu Gly Lys Glu Thr Val Gln Asn Ile LeuAla Leu Arg Phe Ala 180 185 190 Asn Gln Leu Phe Glu Pro Leu Trp Asn SerAsn Tyr Val Asp His Val 195 200 205 Gln Ile Thr Met Ala Glu Asp Ile GlyLeu Gly Gly Arg Ala Gly Tyr 210 215 220 Tyr Asp Gly Ile Gly Ala Ala ArgAsp Val Ile Gln Asn His Leu Ile 225 230 235 240 Gln Leu Leu Ala Leu ValAla Met Glu Glu Pro Ile Ser Phe Val Pro 245 250 255 Ala Gln Leu Gln AlaGlu Lys Ile Lys Val Leu Ser Ala Thr Lys Pro 260 265 270 Cys Tyr Pro LeuAsp Lys Thr Ser Ala Arg Gly Gln Tyr Ala Ala Gly 275 280 285 Trp Gln GlySer Glu Leu Val Lys Gly Leu Arg Glu Glu Asp Gly Phe 290 295 300 Asn ProGlu Ser Thr Thr Glu Thr Phe Ala Ala Cys Thr Leu Glu Ile 305 310 315 320Thr Ser Arg Arg Trp Ala Gly Val Pro Phe Tyr Leu Arg Thr Gly Lys 325 330335 Arg Leu Gly Arg Arg Val Thr Glu Ile Ala Val Val Phe Lys Asp Ala 340345 350 Pro His Gln Pro Phe Asp Gly Asp Met Thr Val Ser Leu Gly Gln Asn355 360 365 Ala Ile Val Ile Arg Val Gln Pro Asp Glu Gly Val Leu Ile ArgPhe 370 375 380 Gly Ser Lys Val Pro Gly Ser Ala Met Glu Val Arg Asp ValAsn Met 385 390 395 400 Asp Phe Ser Tyr Ser Glu Ser Phe Thr Glu Glu SerPro Glu Ala Tyr 405 410 415 Glu Arg Leu Ile Leu Asp Ala Leu Leu Asp GluSer Ser Leu Phe Pro 420 425 430 Thr Asn Glu Glu Val Glu Leu Ser Trp LysIle Leu Asp Pro Ile Leu 435 440 445 Glu Ala Trp Asp Ala Asp Gly Glu ProGlu Asp Tyr Pro Ala Gly Thr 450 455 460 Trp Gly Pro Lys Ser Ala Asp GluMet Leu Ser Arg Asn Gly His Thr 465 470 475 480 Trp Arg Arg Pro 3 29 DNAArtificial Sequence Description of Artificial Sequence syntheticoligomer 3 gatccgatga ggctttggct ctgcgtggc 29 4 29 DNA ArtificialSequence Description of Artificial Sequence synthetic oligomer 4cttcattggt ggactcggta actgcagcg 29 5 37 DNA Artificial SequenceDescription of Artificial Sequence synthetic oligomer 5 aattcgcggccgctctagac tgcagcggcc gcgcatg 37 6 29 DNA Artificial SequenceDescription of Artificial Sequence synthetic oligomer 6 cgcggccgctgcagtctaga gcggccgcg 29 7 28 DNA Artificial Sequence Description ofArtificial Sequence Synthetic DNA 7 aaaaagatct cgacggatcg ttccactg 28 817 DNA Artificial Sequence Description of Artificial Sequence SyntheticDNA 8 gtaaaacgac ggccatg 17 9 35 DNA Artificial Sequence Description ofArtificial Sequence Synthetic DNA 9 cgagtcgact cgcgaagtag cacctgtcacttttg 35 10 33 DNA Artificial Sequence Description of ArtificialSequence Synthetic DNA 10 tggggatccg caccaacaac tgcgatggtg gtc 33 111452 DNA Corynebacterium glutamicum CDS (1)..(1452) 11 atg gtg atc ttcggt gtc act ggc gac ttg gct cga aag aag ctg ctc 48 Met Val Ile Phe GlyVal Thr Gly Asp Leu Ala Arg Lys Lys Leu Leu 1 5 10 15 ccc gcc att tatgat cta gca aac cgc gga ttg ctg ccc cca gga ttc 96 Pro Ala Ile Tyr AspLeu Ala Asn Arg Gly Leu Leu Pro Pro Gly Phe 20 25 30 tcg ttg gta ggt tacggc cgc cgc gaa tgg tcc aaa gaa gac ttt gaa 144 Ser Leu Val Gly Tyr GlyArg Arg Glu Trp Ser Lys Glu Asp Phe Glu 35 40 45 aaa tac gta cgc gat gccgca agt gct ggt gct cgt acg gaa ttc cgt 192 Lys Tyr Val Arg Asp Ala AlaSer Ala Gly Ala Arg Thr Glu Phe Arg 50 55 60 gaa aat gtt tgg gag cgc ctcgcc gag ggt atg gaa ttt gtt cgc ggc 240 Glu Asn Val Trp Glu Arg Leu AlaGlu Gly Met Glu Phe Val Arg Gly 65 70 75 80 aac ttt gat gat gat gca gctttc gac aac ctc gct gca aca ctc aag 288 Asn Phe Asp Asp Asp Ala Ala PheAsp Asn Leu Ala Ala Thr Leu Lys 85 90 95 cgc atc gac aaa acc cgc ggc accgcc ggc aac tgg gct tac tac ctg 336 Arg Ile Asp Lys Thr Arg Gly Thr AlaGly Asn Trp Ala Tyr Tyr Leu 100 105 110 tcc att cca cca gat tcc ttc acagcg gtc tgc cac cag ctg gag cgt 384 Ser Ile Pro Pro Asp Ser Phe Thr AlaVal Cys His Gln Leu Glu Arg 115 120 125 tcc ggc atg gct gaa tcc acc gaagaa gca tgg cgc cgc gtg atc atc 432 Ser Gly Met Ala Glu Ser Thr Glu GluAla Trp Arg Arg Val Ile Ile 130 135 140 gag aag cct ttc ggc cac aac ctcgaa tcc gca cac gag ctc aac cag 480 Glu Lys Pro Phe Gly His Asn Leu GluSer Ala His Glu Leu Asn Gln 145 150 155 160 ctg gtc aac gca gtc ttc ccagaa tct tct gtg ttc cgc atc gac cac 528 Leu Val Asn Ala Val Phe Pro GluSer Ser Val Phe Arg Ile Asp His 165 170 175 tat ttg ggc aag gaa aca gttcaa aac atc ctg gct ctg cgt ttt gct 576 Tyr Leu Gly Lys Glu Thr Val GlnAsn Ile Leu Ala Leu Arg Phe Ala 180 185 190 aac cag ctg ttt gag cca ctgtgg aac tcc aac tac gtt gac cac gtc 624 Asn Gln Leu Phe Glu Pro Leu TrpAsn Ser Asn Tyr Val Asp His Val 195 200 205 cag atc acc atg act gaa gatatt ggc ttg ggt gga cgt gct ggt tac 672 Gln Ile Thr Met Thr Glu Asp IleGly Leu Gly Gly Arg Ala Gly Tyr 210 215 220 tac gac ggc atc ggc gca gcccgc gac gtc atc cag aac cac ctg atc 720 Tyr Asp Gly Ile Gly Ala Ala ArgAsp Val Ile Gln Asn His Leu Ile 225 230 235 240 cag ctc ttg gct ctg gttgcc atg gaa gaa cca att tct ttc gtg cca 768 Gln Leu Leu Ala Leu Val AlaMet Glu Glu Pro Ile Ser Phe Val Pro 245 250 255 gcg cag ctg cag gca gaaaag atc aag gtg ctc tct gcg aca aag ccg 816 Ala Gln Leu Gln Ala Glu LysIle Lys Val Leu Ser Ala Thr Lys Pro 260 265 270 tgc tac cca ttg gat aaaacc tcc gct cgt ggt cag tac gct gcc ggt 864 Cys Tyr Pro Leu Asp Lys ThrSer Ala Arg Gly Gln Tyr Ala Ala Gly 275 280 285 tgg cag ggc tct gag ttagtc aag gga ctt cgc gaa gaa gat ggc ttc 912 Trp Gln Gly Ser Glu Leu ValLys Gly Leu Arg Glu Glu Asp Gly Phe 290 295 300 aac cct gag tcc acc actgag act ttt gcg gct tgt acc tta gag atc 960 Asn Pro Glu Ser Thr Thr GluThr Phe Ala Ala Cys Thr Leu Glu Ile 305 310 315 320 acg tct cgt cgc tgggct ggt gtg ccg ttc tac ctg cgc acc ggt aag 1008 Thr Ser Arg Arg Trp AlaGly Val Pro Phe Tyr Leu Arg Thr Gly Lys 325 330 335 cgt ctt ggt cgc cgtgtt act gag att gcc gtg gtg ttt aaa gac gca 1056 Arg Leu Gly Arg Arg ValThr Glu Ile Ala Val Val Phe Lys Asp Ala 340 345 350 cca cac cag cct ttcgac ggc gac atg act gta tcc ctt ggc caa aac 1104 Pro His Gln Pro Phe AspGly Asp Met Thr Val Ser Leu Gly Gln Asn 355 360 365 gcc atc gtg att cgcgtg cag cct gat gaa ggt gtg ctc atc cgc ttc 1152 Ala Ile Val Ile Arg ValGln Pro Asp Glu Gly Val Leu Ile Arg Phe 370 375 380 ggt tcc aag gtt ccaggt tct gcc atg gaa gtc cgt gac gtc aac atg 1200 Gly Ser Lys Val Pro GlySer Ala Met Glu Val Arg Asp Val Asn Met 385 390 395 400 gac ttc tcc tactca gaa tcc ttc act gaa gaa tca cct gaa gca tac 1248 Asp Phe Ser Tyr SerGlu Ser Phe Thr Glu Glu Ser Pro Glu Ala Tyr 405 410 415 gag cgc ctc attttg gat gcg ctg tta gat gaa tcc agc ctc ttc cct 1296 Glu Arg Leu Ile LeuAsp Ala Leu Leu Asp Glu Ser Ser Leu Phe Pro 420 425 430 acc aac gag gaagtg gaa ctg agc tgg aag att ctg gat cca att ctt 1344 Thr Asn Glu Glu ValGlu Leu Ser Trp Lys Ile Leu Asp Pro Ile Leu 435 440 445 gaa gca tgg gatgcc gat gga gaa cca gag gat tac cca gcg ggt acg 1392 Glu Ala Trp Asp AlaAsp Gly Glu Pro Glu Asp Tyr Pro Ala Gly Thr 450 455 460 tgg ggt cca aagagc gct gat gaa atg ctt tcc cgc aac ggt cac acc 1440 Trp Gly Pro Lys SerAla Asp Glu Met Leu Ser Arg Asn Gly His Thr 465 470 475 480 tgg cgc aggcca 1452 Trp Arg Arg Pro 12 484 PRT Corynebacterium glutamicum 12 MetVal Ile Phe Gly Val Thr Gly Asp Leu Ala Arg Lys Lys Leu Leu 1 5 10 15Pro Ala Ile Tyr Asp Leu Ala Asn Arg Gly Leu Leu Pro Pro Gly Phe 20 25 30Ser Leu Val Gly Tyr Gly Arg Arg Glu Trp Ser Lys Glu Asp Phe Glu 35 40 45Lys Tyr Val Arg Asp Ala Ala Ser Ala Gly Ala Arg Thr Glu Phe Arg 50 55 60Glu Asn Val Trp Glu Arg Leu Ala Glu Gly Met Glu Phe Val Arg Gly 65 70 7580 Asn Phe Asp Asp Asp Ala Ala Phe Asp Asn Leu Ala Ala Thr Leu Lys 85 9095 Arg Ile Asp Lys Thr Arg Gly Thr Ala Gly Asn Trp Ala Tyr Tyr Leu 100105 110 Ser Ile Pro Pro Asp Ser Phe Thr Ala Val Cys His Gln Leu Glu Arg115 120 125 Ser Gly Met Ala Glu Ser Thr Glu Glu Ala Trp Arg Arg Val IleIle 130 135 140 Glu Lys Pro Phe Gly His Asn Leu Glu Ser Ala His Glu LeuAsn Gln 145 150 155 160 Leu Val Asn Ala Val Phe Pro Glu Ser Ser Val PheArg Ile Asp His 165 170 175 Tyr Leu Gly Lys Glu Thr Val Gln Asn Ile LeuAla Leu Arg Phe Ala 180 185 190 Asn Gln Leu Phe Glu Pro Leu Trp Asn SerAsn Tyr Val Asp His Val 195 200 205 Gln Ile Thr Met Thr Glu Asp Ile GlyLeu Gly Gly Arg Ala Gly Tyr 210 215 220 Tyr Asp Gly Ile Gly Ala Ala ArgAsp Val Ile Gln Asn His Leu Ile 225 230 235 240 Gln Leu Leu Ala Leu ValAla Met Glu Glu Pro Ile Ser Phe Val Pro 245 250 255 Ala Gln Leu Gln AlaGlu Lys Ile Lys Val Leu Ser Ala Thr Lys Pro 260 265 270 Cys Tyr Pro LeuAsp Lys Thr Ser Ala Arg Gly Gln Tyr Ala Ala Gly 275 280 285 Trp Gln GlySer Glu Leu Val Lys Gly Leu Arg Glu Glu Asp Gly Phe 290 295 300 Asn ProGlu Ser Thr Thr Glu Thr Phe Ala Ala Cys Thr Leu Glu Ile 305 310 315 320Thr Ser Arg Arg Trp Ala Gly Val Pro Phe Tyr Leu Arg Thr Gly Lys 325 330335 Arg Leu Gly Arg Arg Val Thr Glu Ile Ala Val Val Phe Lys Asp Ala 340345 350 Pro His Gln Pro Phe Asp Gly Asp Met Thr Val Ser Leu Gly Gln Asn355 360 365 Ala Ile Val Ile Arg Val Gln Pro Asp Glu Gly Val Leu Ile ArgPhe 370 375 380 Gly Ser Lys Val Pro Gly Ser Ala Met Glu Val Arg Asp ValAsn Met 385 390 395 400 Asp Phe Ser Tyr Ser Glu Ser Phe Thr Glu Glu SerPro Glu Ala Tyr 405 410 415 Glu Arg Leu Ile Leu Asp Ala Leu Leu Asp GluSer Ser Leu Phe Pro 420 425 430 Thr Asn Glu Glu Val Glu Leu Ser Trp LysIle Leu Asp Pro Ile Leu 435 440 445 Glu Ala Trp Asp Ala Asp Gly Glu ProGlu Asp Tyr Pro Ala Gly Thr 450 455 460 Trp Gly Pro Lys Ser Ala Asp GluMet Leu Ser Arg Asn Gly His Thr 465 470 475 480 Trp Arg Arg Pro 113

1. A polypeptide which comprises the amino acid sequence represented bySEQ ID NO:2.
 2. A polypeptide which comprises an amino acid sequence inwhich Ala at position 213 in the amino acid sequence represented by SEQID NO:2 is replaced with an other amino acid, and hasglucose-6-phosphate dehyrdogenase activity.
 3. A polypeptide whichcomprises the amino acid sequence represented by SEQ ID NO:12.
 4. Apolypeptide which comprises an amino acid sequence in which oneor-several amino acids other than the amino acid residue at position 213in the amino acid sequence of the polypeptide according to claim 2 aredeleted, substituted or added, and has glucose-6-phosphate dehyrdogenaseactivity.
 5. A polypeptide which comprises an amino acid sequence inwhich one or several amino acids other than the amino acid residue atposition 213 in the amino acid sequence represented by SEQ ID NO:12 aredeleted, substituted or added, and has glucose-6-phosphate dehyrdogenaseactivity.
 6. A DNA which encodes the polypeptides according to any oneof claims 1 to
 5. 7. A DNA which comprises the nucleotide sequencerepresented by SEQ ID NO:1.
 8. A DNA which comprises a nucleotidesequence in which a nucleotide sequence of positions 637 to 639 encodingAla in the nucleotide sequence represented by SEQ ID NO:1 is replacedwith a codon encoding an amino acid other than Ala.
 9. A DNA whichcomprises the nucleotide sequence represented by SEQ ID NO:11.
 10. A DNAwhich hybridizes with a DNA comprising the nucleotide sequencerepresented by SEQ ID NO:1 under stringent conditions, and encodes apolypeptide having glucose-6-phosphate dehydrogenase activity, wherein anucleotide sequence corresponding to the nucleotide sequence ofpositions 637 to 639 encoding Ala in the nucleotide sequence representedby SEQ ID NO:1 is replaced with a codon encoding an amino acid otherthan Ala.
 11. A DNA which hybridizes with a DNA comprising thenucleotide sequence represented by SEQ ID NO:1 under stringentconditions, and encodes a polypeptide having glucose-6-phosphatedehyrdogenase activity, wherein a nucleotide sequence corresponding tothe nucleotide of position 637 in the nucleotide sequence represented bySEQ ID NO:1 is replaced with adenine.
 12. A recombinant DNA which isobtainable by inserting the DNA according to any one of claims 6 to 11into a vector.
 13. The recombinant DNA according to claim 12, whereinthe recombinant DNA is replicable in a microorganism belonging to thegenus Escherichia or the genus Corynebacterium.
 14. A plasmid pCRBzwfMcomprised in Escherichia coli TOP10 (FERM BP-7135).
 15. A transformantwhich is obtainable by introducing the recombinant DNA or plasmidaccording to any one of claim 12 to 14 into a host cell.
 16. Thetransformant according to claim 15, wherein the host cell is amicroorganism which is capable of producing L-amino acid.
 17. Thetransformant according to claim 16, wherein the host cell is amicroorganism belonging to the genus Escherichia or the genusCorynebacterium.
 18. A transformant belonging to the genus Escherichiaor the genus Corynebacterium, which comprises a chromosome into whichthe DNA according to any one of claims 6 to 11 is artificiallyintegrated.
 19. The transformant according to claim 17 or 18, whereinthe microorganism belonging to the genus Corynebacterium isCorynebacterium gIutamicum.
 20. A process for producing a polypeptide,which comprises culturing the transformant according to any one ofclaims 15 to 19 in a medium to form and accumulate the polypeptideaccording to any one of claims 1 to 5 in a culture, and recovering thepolypeptide from the culture.
 21. A process for producing L-amino acid,which comprises culturing the transformant according to any one ofclaims 16 to 19 in a medium to form and accumulate L-amino acid which isbiosynthesized using NADPH in the culture, and recovering the L-aminoacid from the culture.
 22. The process for producing L-amino acidaccording to claim 21, wherein the L-amino acid which is biosynthesizedusing NADPH is selected from L-lysine, L-threonine, L-isoleucine,L-tryptophan, L-phenylalanine, L-tyrosine, L-histidine and L-cysteine.23. The process for producing L-amino acid according to claim 21,wherein the L-amino acid is L-lysine.